Beta-glucosidases comprise members of Glycosyl Hydrolase Families 1 and 3 whose primary enzymatic function is the hydrolysis of the beta-glycosidic bonds linking carbohydrate residues in cellobiose or soluble cellodextrins. Some beta-glucosidases are specific for cellobiose or aryl glucosides, most of those characterized have broad specificity and can hydrolyse a broad range of substrates (Bhatia et al., 2002). Under certain conditions, these enzymes can also catalyze the synthesis of glycosidic linkages through the reverse reaction or through transglycosylation (Sinnott, 1990). Both the hydrolytic and synthetic capabilities of this enzyme class can be employed in biotechnological applications.
One major application of the hydrolytic activity of beta-glucosidase is the alleviation of product inhibition of cellulase systems. Cellobiose, a major product of cellulose hydrolysis, strongly inhibits the activity of cellobiohydrolases (EC 3.2.1.91). The inclusion of sufficient beta-glucosidase to hydrolyse cellobiose to glucose, which is less inhibitory, results in significant gains in activity at higher degrees of substrate conversion (U.S. Pat. No. 6,015,703).
Numerous other biotechnological applications of beta-glucosidase have been reviewed by Bhatia et al. (2002). Examples of applications that depend on its hydrolytic activity include the release of medically important compounds from flavanoid and isoflavanoid glucosides and the liberation of fragrant compounds in fruit juices and wines (U.S. Pat. No. 6,087,131). A representative use of the synthetic activity is the production of alkyl-glucosides for use as a detergent (Ducret et al., 2002).
The activity of glycosyl hydrolases, including beta-glucosidases, is dependent on specific protonation states of the catalytic amino acids (glutamate or aspartate) in the active site of the enzyme, which are influenced by pH. For example, beta-glucosidase I from Trichoderma reesei is most active in the pH range 5.0-5.5 and dramatically less so under more acidic or basic conditions (Woodward and Arnold, 1981).
The pH dependencies of the activity and stability of a protein are not necessarily related. The destabilizing effects of acidic or alkaline conditions result from protonation or deprotonation of amino acid sidechains which may not be catalytic or even in close proximity to the active site. pH-dependent denaturation primarily results from the differing pKa values of specific amino acid sidechains in the native and denatured states, which introduce pH dependence to the free energy difference between states. Additionally, proteins can become highly charged at extremes of pH and experience increased intramolecular electrostatic repulsion (Fersht, 1998).
An engineered enzyme with an altered pH optimum will not necessarily be stable at the new pH for extended periods. Several glycoside hydrolases have undergone mutagenesis to alter their activity or stability at pH values important for industrial applications. For a starch processing application, an alpha-amylase from Bacillus licheniformis was engineered to include two amino acid replacements, MIST and N188S, which increased its low pH (5.2) activity to 140% of wild-type (U.S. Pat. No. 5,958,739). The addition of a third mutation, H133Y, further increased the activity to more than 150% of the double mutant. Replacement of a loop in a Bacillus endoglucanase, identified with a rational design approach, shifted its pH optimum for a textiles application. With an Ala-Gly-Ala replacement, the pH optimum was shifted up by more than 1 pH unit (U.S. Publication No. 2005/0287656). A further example is the incorporation of amino acid replacements A162P and W62E into Ce145 endoglucanase from Humicola insolens. These mutations increased activity at alkaline conditions (pH 10) to 124-144% of that of the wild-type (U.S. Pat. No. 5,792,641). As a final example, the activity of a recombinant Trichoderma xylanase at pH above 5.5 was significantly improved by incorporation of various combinations of the replacements N10H, N11D, Y27M, and N29L (U.S. Pat. No. 5,866,408).
There are few reports of engineering beta-glucosidases though mutagenesis to modulate properties of these enzymes. For example, the thermostability of a quadruple mutant A16T/G142S/H226Q/D703G of an Aspergillus beta-glucosidase was increased such that it retained ˜50% of its activity after one hour of incubation at 65° C. vs. 0-5% for wild-type variants. This enzyme was constructed using a combination of random mutagenesis, site-saturation and shuffling (U.S. Publication No. 2004/013401). Amino acid substitutions at one or more of positions 43, 101, 260 and 543 of Trichoderma reesei beta-glucosidase I resulted in modified beta-glucosidase with increased catalytic efficiency (U.S. Provisional Application No. 61/182,275). The following mutations were found to be particularly advantageous for increasing the catalytic efficiency of Trichoderma reesei beta-glucosidase I: V43I, V43C, V101A, V101G, F260I, F260V, F260Q, F260D, I543N, I543W, I543A, I543S, I543G, and I543L.
It is also noted that enzymes with altered pH stability profiles do not necessarily require altered pH optima to be of utility. For example, the pH of a cell culture used to express an enzyme may be different than the intended working pH of the enzyme in its application as a biocatalyst. If the stability of the enzyme is compromised at the expression pH, the overall yield of enzymatic activity will be reduced. This has been observed with Trichoderma reesei beta-glucosidase expressed in Saccharomyces cerevisiae (Cummings and Fowler, 1996). An unbuffered expression medium was observed to become more acidic over time, dropping from pH 6.0 to 2.0-3.0; this pH drop was correlated to a sharp decline in beta-glucosidase activity.
Instability can be further exacerbated by hydrodynamic shear arising from mixing of the cell culture, particularly in the presence of gas-liquid interfaces such as those produced by aeration (Weijers and Van't Riet, 1992; Elias and Joshi, 1998). Some enzymes may be further inactivated by shear stresses present during post-production processes such as ultrafiltration or in their final applications.
Shear inactivation of glycosyl hydrolases has been reported in the literature: Jones and Lee (1988) described the inactivation of a T. reesei cellulase mixture in a reactor system incorporating a high speed impeller, but only in the presence of air; Sachse et al. (1990) reported a higher specific activity of T. reesei cellulase produced in a low-shear vs. a conventional stirred reactor; Reese (1980) also reported the inactivation of T. reesei cellulase by shaking during hydrolysis and observed that the effect could be ameliorated by the use of surfactants; finally, Gunjikar et al. (2001) reported deactivation of exoglucanases, endoglucanases and beta-glucosidase in mixed reactors, the magnitude of which was proportionate to the mixing energy applied.